Characterization of the Porcine CLEC12A and Analysis of Its Expression on Blood Dendritic Cell Subsets

Abstract

CLEC12A has been proposed as a suitable target for delivering antigen to dendritic cells (DCs) to enhance vaccine efficacy both in human and mouse. In this study, we have characterized the porcine homolog of CLEC12A (poCLEC12A). Using new monoclonal antibodies (mAb), raised against its ectodomain, poCLEC12A was found to be expressed on alveolar macrophages, blood conventional type 1 and type 2 DCs and plasmacytoid DCs, but not on monocytes, T cells, B cells or NK cells, in contrast to its human and murine homologs. Western blot analysis showed that in alveolar macrophages this receptor is expressed both as a monomer and a dimer. After binding to DCs, anti- poCLEC12A mAb was efficiently internalized. No significant changes were observed in TNFα or IFNα secretion by plasmacytoid DCs stimulated with either CpGs (ODN2216) or polyinosinic-polycytidylic acid (poly I:C), upon incubation with mAb. These results provide the basis for future investigations aimed to assess the ability of anti-poCLEC12A mAbs to improve vaccine efficacy by targeting antigen to DCs.

Keywords: C-type lectin; CLEC12A; dendritic cell; monoclonal antibody; pig.

Figure 1

Reactivity of mAbs FA6A5 and FA2B10 with poCLEC12A transfectants. CHO cells transiently transfected with poCLEC12A-Nt-GFP construct were stained with mAbs FA6A5, FA2B10, or an IgG1 control antibody followed by APC-goat F(ab')2 anti-mouse Igs and analyzed by flow cytometry. A control staining of non-transfected CHO cells is also shown.

Figure 2

Expression of CLEC12A on porcine leukocyte subsets. (A) Flow cytometric analysis of porcine alveolar macrophages stained with biotin-conjugated mAb FA2B10 or FA6A5 followed by streptavidin BV421. Open histograms correspond to the negative control using an irrelevant isotype-matched mAb. (B) PBMC were double stained with anti-CD3, anti-CD4, anti-CD8α, anti-CD21 or anti-CD172a mAbs, and PE-conjugated rabbit F(ab')2 anti-mouse Ig (x-axis), followed by biotin-conjugated FA2B10 and streptavidin BV421 (y-axis). Isotype-matched irrelevant mAbs, unlabeled or biotin-labeled were used as negative controls. For analysis, cells were gated as FSC^hi (region R) to enrich the cDC population. Numbers indicate the percentage of cells within the respective quadrants. Results are representative of three independent experiments.

Figure 3

Expression of CLEC12A on plasmacytoid dendritic cells (pDCs). PBMC were first stained with anti-CD172a mAb BA1C11 and anti-CD4 mAb 74-12-4 followed by APC-conjugated goat anti-mouse IgG1 and PE-conjugated goat anti-mouse IgG2b. After blocking free binding sites, cells were incubated with biotin-labeled anti-CLEC12A mAb FA2B10 and streptavidin BV421. After doublet exclusion, cells with high FSC and SSC were selected and pDCs were identified as the CD172a^lo CD4^hi subpopulation. CLEC12A expression in gated population is shown in the filled histogram; open histogram corresponds to the negative control with an irrelevant isotype-matched mAb. The profiles shown are from a representative experiment out of three performed with cells from different donors.

Figure 4

CLEC12A expression on blood conventional dendritic cells (cDCs). PBMC were first incubated with anti-CADM1 (3E1), anti-CD172a (74-22-15) and biotin-labeled anti-CLEC12A (FA2B10) mAbs followed by PE-conjugated goat anti-mouse IgG2b and Alexa Fluor 647-conjugated goat anti-chicken IgY. After blocking free binding sites, cells were incubated with FITC-conjugated anti-CD14 mAb MIL-2 and streptavidin BV421. Cells with high FSC and SSC were selected. Among these cells, cDC1 were gated as CD14⁻ CD172a^lo CADM1⁺, cDC2 as CD14⁻ CD172a^hi CADM1⁺, and monocytes as CD14⁺. Expression of CLEC12A in gated populations is shown as filled histograms; open histograms correspond to negative controls using an irrelevant isotype-matched mAb. The profiles shown are from a representative experiment out of three performed with cells from different donors.

Figure 5

Molecular characterization of porcine CLEC12A. Lysates from alveolar macrophages were resolved by 7.5% SDS-PAGE under reducing (A) or non-reducing conditions (B), transferred to nitrocellulose membranes and probed with mAb FA6A5. Numbers in the right indicate position and size of MW markers. Results are representative of two independent experiments.

Figure 6

Effect of poCLEC12A engagement on the production of cytokines. FACS-sorted pDCs were cultured with anti-poCLEC12A mAb FA2B10, an isotype-matched control mAb or in medium alone, in the presence of either CpG/ODN2216 (5 μg/ml) (A) or poly I:C (10 μg/ml) (B) for 18 h, after which supernatants were collected and analyzed by ELISA for cytokine production. Results shown (mean + SD) are from a representative experiment, run in triplicates (number of performed experiments: 3 for CpG, 2 for poly I:C, performed with cells from different donors).

Figure 7

Binding of mAb FA2B10 to CLEC12A induces receptor internalization in blood DC subsets. (A) PBMCs were labeled with biotin-conjugated anti-CLEC12A mAb FA2B10, followed by 0- to 60-min incubation at 37°C. Surface expression of bound anti-CLEC12A mAb was analyzed by flow cytometry following labeling with streptavidin BV421. cDC subsets were identified according to the expression of CD14, CD172a, and CADM-1 markers; pDCs were identified based on the expression of CD4. Results are representative of three independent experiments. (B) CLEC12A internalization was calculated relative to the surface expression in cells kept at 4°C based on the mean fluorescence intensity. Data are shown as mean+SD of three independent experiments. (C) Analysis by confocal microscopy of CLEC12A internalization in pDCs. Cells were stained with biotinylated anti-CLEC12A mAb FA2B10, followed by labeling with Alexa Fluor 488-conjugated streptavidin (green). Then, cells were incubated at either 37°C, for allowing internalization, or 4°C, to prevent it, and subsequently stained with anti-CD4 mAb 74-12-4, and Alexa Fluor 594-conjugated anti-mouse IgG2b (red). Yellow arrowheads show anti-CLEC12A green labeled complexes inside cell; bluish arrowheads point to surface located anti-CLEC12A.